Isolated and purified nucleic acids comprising a gene and a regulatory region for the gene expression of the same

ABSTRACT

An isolated and purified nucleic acid comprising a gene specifically expressed in hop lupulin glands. Hops are dioecious, and only female plants bear cones, the lupulin glands of which contain secondary metabolic products which provide bitterness and flavor to beer. These secondary metabolic products contain some pharmacologically effective compounds. In order to breed a more useful cultivar of hops by manipulating the constituent of such useful secondary metabolic products relying on genetic engineering techniques, this invention provides an isolated and purified nucleic acid comprising a gene specifically expressed in hop lupulin glands. By using this nucleic acid, it is possible to develop a novel method for breeding hops with transformation techniques and molecular selection techniques. Furthermore, the present invention also provides a nucleic acid comprising the regulatory region for specifically expressing genes in lupulin glands. This nucleic acid can be used also for hop breeding.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to nucleic acids comprising a genespecifically expressed in lupulin glands of hops and to regulatorysequences thereof.

[0003] 2. Discussion of the Background

[0004] Plants produce and store a wide variety of low molecular weightorganic compounds including terpenoids, alkaloids, phenolics, saponins,etc. Since, formerly, these compounds were not considered to be directlyinvolved in supporting living matter having only minor biologicalfunctions, they were conventionally called “secondary metabolicproducts”.

[0005] Now, however, it has been elucidated that these secondarymetabolic products function for promoting cellular differentiation andprotecting cells from external harmful factors, and, furthermore, thesesecondary metabolic products formed by plants have been utilized andapplied in a wide field of popular foods, medicaments, dyes, etc.

[0006] These secondary metabolic products have been paid so muchattention with respect to their usefulness that the production pathwaysthereof in plant cells have been successively elucidated, indicatingthat these compounds are produced via a complicated biosynthetic cascadeinvolving a number of enzymes. Most of these compounds biosynthesizedvia a cascade of enzymatic reactions can be isolated by directlyextracting plant materials, but such a direct extraction from plants notonly does not meet the demand for production on a large scale, but alsois generally expensive. Therefore, the development of methods forsynthesizing these compound in vitro using cultured cells, etc. has beenunder way.

[0007] On the one hand, it has been elucidated that hops, a majormaterial for rendering a refreshing bitterness and flavor to beer,secrete a variety of secondary metabolic products in lupulin glands onthe cones, contributing a great deal to the bitterness and flavor ofbeer.

[0008] Based on these circumstances, hops have been subjected to variousbreeding attempts focused on secondary metabolic products accumulated inlupulin glands such as bitter substance, essential oils, etc. inaddition to the improvement of their agricultural properties.

[0009] However, hops are a dioecious plant, and especially the maleplant bears no cones, materials for beer, is not commerciallyappreciated, and accordingly has not been actively studied so that itsgenetic properties useful for beer brewing have hardly been elucidatedat all. Therefore, at present, hop breeding by conventional crossingrelies largely on breeders' experience and intuition, and no predictioncan be made especially on the quality of fermentation products at alltill the time of the actual bearing of cones.

[0010] On the other hand, these days, breeding methods using geneticengineering such as a transformation technique and marker assistedselection have become available for various plants. In these methods, amore objective breeding can be performed compared with thoseconventional breeding methods that largely depend on breeders'experience and intuition. The transformation technique is a techniquefor directly introducing a desired character by transferring andexpressing a foreign gene in plant cells. The expression of a foreigngene can be performed by linking a desired structural gene and aterminator capable of functioning in plant cells to a gene expressionregulating promoter which is capable of functioning also in plant cells,and transferring the resulting transformed promoter into plant cells.Promoters frequently used in experiments are exemplified by CaMV 35Scapable of expressing a transferred gene in regardless of any tissues ofrelatively numerous varieties of plants, and the promoter for thenopaline synthetase gene (Sanders, P. R., et al., Nucleic Acid Res., 15(1987) 1543-1558). Furthermore, in the practical aspect of genetictransformation, the transferred gene might be harmful for the plantgrowth, etc. Therefore, there has been also a demand for promoterscapable of expressing a foreign gene in a desired tissue, desiredperiod, and desired quantity. The advantage of the breeding method usingthe transformation technique over the conventional traditional breedingmethod is that the former method is capable of transducing a desiredcharacter to plants regardless of their species with a relatively highaccuracy in a short time. Also in the case of hops, since they can beproliferated by root-planting, the procedure for fixing the transducedcharacter is not required. Therefore, the breeding method using thetransformation technique is especially effective for hops.

[0011] Marker assisted selection is an example of a breeding methodusing the DNA marker such as RFLP (Restriction Fragment LengthPolymorphism) marker, and have been put into practical use, especiallyfor rice and wheat. It has been generally agreed that transformationtechniques are capable of transducing a character regulated by a singlegene, but incapable of transducing a character regulated by multiplegenes. Marker assisted selection is capable of compensating for thesedefects of transformation techniques.

[0012] A prerequisite for such a breeding method using gene technologyis to elucidate genes related with the desired character and thoseregulating those genes. Especially, from the viewpoint of hops as thebeer material as well as the source of effective drug ingredient, ifgenes related to the biosynthesis of secondary metabolic productssecreted from lupulin glands contained in the cones of female plants areelucidated, these genes can be applied to the hop breeding method usingthe gene technology, and, furthermore, also to the field of medicaltreatment.

SUMMARY OF THE INVENTION

[0013] Therefore, in order to elucidate genes specifically expressed inlupulin glands and facilitate their practical application, it is anobject of the present invention to isolate, purify and provide suchgenes, as well as regulatory sequences thereof, such as promoters, forthese genes.

[0014] As described above, nucleic acids isolated and purified in thepresent invention comprise genes specifically expressed in the lupulinglands of hops, promoters specifically functioning in lupulin glands,and portions thereof.

[0015] Using these nucleic acids, the conventional method for breedinghops wholly dependent on the breeders experience and intuition can beconverted to a more objective method using genetic engineering. Asdescribed above, since important secondary metabolic products, such asbeer materials and effective drug ingredients, are secreted exclusivelyin lupulin glands on the hop cones, genes specifically expressed tofunction in lupulin glands are likely related to the biosynthesis ofthese secondary metabolic products. Thus, by utilizing genes capable ofparticipating in the biosynthesis of secondary metabolic products as amarker, an improved marker assisted selection can be developed for thebreeding of hops which will contribute significantly to the food anddrug industries. In addition, by transferring the above-described genesto hops using a transformation technique, breeding of industriallyuseful cultivar can be accomplished. That is, by breeding hops using agenetic engineering technique with these nucleic acids, the compositionof secondary metabolic products accumulating in lupulin glands can beregulated. Furthermore, the nucleic acids of the present inventionenable the maintenance and improvement of hop quality for beer brewing,and the use of hops for drug production.

[0016] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1: diagram representing the procedure of inverse PCR in thecase of isolation of the regulatory sequence in the lupulin-specificgene.

[0018]FIG. 2: results of Northern blot analysis of RNAs which have beenrecovered from lupulin-rich fraction and lupulin-poor fraction andelectrophoresed using a lupulin-specific gene as the probe. Theanalytical results indicate the specificity of lupulin-specific geneexpression in lupulin glands.

DETAILED DESCRIPTION OF THE INVENTION

[0019] By the above-described expression “specifically expressed” or“specifically functions,” it is meant that the genes are expressed orfunctioning not only in lupulin glands alone but also doing so more inthese glands as compared to other organs. That is, whether the genes areexpressed or functioning “specifically” in lupulin glands or not can bedetermined by their expression amount and function intensity in lupulinglands compared with other organs.

[0020] Furthermore, by the expression “specifically expressed” or“specifically functions” it is not only meant that the genes are asspecific as defined above throughout the entire developing period, etc.but also that the expression and function of the genes are more highlyelevated by the specific developing period or external influencescompared with other organs.

[0021] The above-described nucleic acids comprise both DNA and RNA.Also, the type of “genes” coded in the above-described nucleic acidsincludes any types such as genomic DNA, cDNA and mRNA.

[0022] Further, portions of the above-described nucleic acids are alsoincluded in the present invention. In some applications, even a partialsequence thereof alone is capable of functioning without a whole lengththereof being required, i.e., use of a fragment having the desiredfunctional property of the full-length sequence. For example, in thecase of application of these nucleic acids to the breeding method by themarker assisted selection based on RFLP, molecules are identified byhybridization and PCR, wherein the size of probes and PCR primers usedis sufficient if they comprise a portion of the above-described specificnucleic acids derived from lupulin glands, for example, a partialcontinuous sequence of several tens to several hundreds bp long.

[0023] Furthermore, in the present invention, the above-described genesspecifically expressed in lupulin glands feature that the genes encodeproteins related to the biosynthesis of secondary metabolic productsgenerated in lupulin glands.

[0024] Proteins herein used include, for example, the amino acidsequence described in SEQ ID NO:1. Genes encoding the protein includethose having the base sequence described in SEQ ID NO:2, and also thosehaving the base sequence partially different from that of SEQ ID NO:2but reserving the very base sequence encoding the above-described aminoacid sequence. In the case of the use of this base sequence as probesand PCR primers, it can be modified to a certain extent so far as theresulting sequence retains the desired functional capability. All aminoacid sequences encoding the above-described amino acid sequences arewithin the scope of the present invention. Specific nucleic acidsequences other than those described above are readily determined byusing the well-established genetic code for codons which encode theamino acid residues of the proteins described above. The genetic code isset forth in L. Stryer, Biochemistry, Third Edition, 1988, W. H. Freemanand Co., incorporated herein by reference in its entirety.

[0025] Also, isolated and purified nucleic acids of the presentinvention comprise the gene encoding chalcone synthetase. This chalconesynthetase is the enzyme related to the metabolism of phenylalanine andtyrosine, and, more specifically, has been determined to catalyze theconversion of 1 mole of coumaroyl CoA and 3 moles of malonyl CoA to4,2,4,6-tetrahydroxychalcone (naringenin-chalcone) in the biosynthesisof flavonoids. Therefore, the above-described nucleic acids can be usedto regulate the metabolic system involved in the biosynthesis offlavonoids in plants, and also as a gene marker for characters relatedto flavonoids. Furthermore, recently, it has been indicated that achalcone synthetase-like enzyme possibly has a valerophenone synthetaseactivity which catalyzes the biosynthesis of phlorisovalerophenone andphlorisobutyrophenone, the precursors of bitter substance, α-acid andβ-acid (European Brewery Convention, Proceedings of the 26th Congress,p. 215 (1997)). These facts indicate that the protein encoded by thegene isolated in the present invention functions as the valerophenonesynthetase participating in the biosynthesis of bitter substance.Accordingly, the above-described nucleic acids can be used for theregulation of the metabolic system concerning the biosynthesis of bittersubstance in hops and also as the gene marker for the character relatedto bitter substance.

[0026] Nucleic acids isolated and purified in the present invention alsoinclude a regulatory sequence for the specific expression of the gene inlupulin glands, and the sequence contains a promoter which is activatedin lupulin glands. Such a sequence includes, for example, one having thebase sequence described in SEQ ID NO:1. This regulatory sequencespecific in lupulin glands can be used to facilitate the expression ofgenes linked downstream thereof in lupulin glands.

[0027] Furthermore, it is an object of the present invention to providea vector containing a gene specifically expressed in lupulin glands or aregulatory sequence specifically regulating the expression of gene inlupulin glands.

[0028] Breeding of plants such as hops can be achieved by transformingplants including hops using a vector bearing a gene specificallyexpressible in the above-described lupulin glands. Especially, such avector becomes effective for the breeding by the elevation/suppressionof the production of secondary metabolic products. Furthermore, thisvector can be used not only for the plant breeding but also theproduction of secondary metabolic products by expressing the generelated to the biosynthesis the secondary metabolic products in culturedcells. If the production of secondary metabolic products becomespossible in cultured cells, the isolation of the secondary metabolicproducts can be easily performed.

[0029] Also, the above-described vector bearing a regulatory sequencecan be used not only for the expression of the specific genes but alsofor the specific expression of a desired gene in hop lupulin glands bylinking a gene derived from hops or different plant species downstreamof the regulatory sequence. By doing so, any desired gene in lupulinglands can be expressed.

[0030] In addition, the present invention also includes plant cellstransformed by the above-described vector. Herein, plant cells caninclude, without any limitations in their morphology or growing stages,various types of cells such as cultured cells, callus, protoplasts,plant, etc. This invention can also include not only plant cells of thefirst generation but also plants generated from the first generationplant cells.

[0031] The above-described transformed plant enables the expression ofdesired genes including those encoding secondary metabolic products bythe transfer of the above-described vector, increasing the usefulness ofplants as materials for foods and drugs.

[0032] In the following description, the present invention will bedescribed in detail with reference to preferred embodiments.

[0033] 1. Isolation of Nucleic Acids Comprising Hop LupulinGland-specific Gene and the Expression Regulatory Sequence Thereof

[0034] (1) Preparation of Total RNA and mRNA

[0035] Total RNA can be prepared by the existing method, for example, amethod described in “Protocols of Plant PCR Experiment”, Shujun-sha, p.56 (1995), incorporated herein by reference. The preparation of mRNAfrom the total RNA can be carried out by the existing method, forexample, according to the protocol attached to “Oligotex-dT30<Super>”available from Takara-Shuzo.

[0036] (2) Preparation of cDNA Library

[0037] cDNA library can be prepared from mRNA by the existing method.cDNA can be prepared, for example, according to the protocol attached to“cDNA synthesis module”, Amersham. Also, the formation of a library ofcDNA thus prepared can be performed according to protocols attached to“cDNA rapid adaptor ligation module” and “cDNA rapid cloning module”both from Amersham, and “GIGAPACK II Plus Packaging Extract”,Stratogene. All of the above-cited publications are incorporated hereinby reference.

[0038] (3) Preparation of Lupulin-specific Probes

[0039] By lupulin-specific probes is meant gene fragments complementaryto genes specifically expressed in lupulin glands. In the presentpreferred embodiment, the lupulin-specific probes can be obtained by thefollowing method.

[0040] Cones approximately 15 days after blooming are divided into afraction comprising mainly lupulin glands and the bracteole base densewith lupulin glands (lupulin-rich fraction) and a fraction comprisingmainly the stipular bract containing few lupulin glands (lupulin-poorfraction), respectively. A group of genes expressed in the lupulin-richfraction has subtracted from it a group of genes also expressed in thelupulin-poor fraction, and a group of remaining genes are considered tobe the ones specifically expressed in lupulin glands with a highprobability.

[0041] Such a subtraction of a group of genes expressed also in thelupulin-poor fraction from a group of genes expressed in thelupulin-rich fraction can be carried out by the existing method,conveniently, for example, according to the protocol attached to a“Subtractor Kit” from Invitrogen.

[0042] (4) Isolation of Lupulin-specific cDNA

[0043] By “lupulin-specific cDNA” is meant cDNA derived from the genespecifically expressed in lupulin glands. Isolation of lupulin-specificcDNA can be performed by screening cDNA library prepared from thelupulin-rich fraction using the lupulin-specific probes. This screeningcan be carried out by the existing method, for example, by a methoddescribed in the “User's guide for performing the hybridization usingDIG system” (Boehringer Mannheim, p. 37 (1995)), incorporated herein byreference.

[0044] Labeling of lupulin-specific probes can be also carried out bythe existing method, for example, according to the protocol attached to“DIG-High Prime”, Boehringer Mannheim.

[0045] (5) Preparation of Hop Genomic DNA

[0046] Preparation of hop genomic DNA can be performed by the existingmethod, for example, a method described in “Protocols for plant PCRexperiment” (Shu-jun Sha, p. 54 (1995)), incorporated herein byreference.

[0047] (6) Isolation of Nucleic Acid Comprising a Regulatory Region forthe Lupulin-specific Gene Expression

[0048] By “nucleic acid comprising a regulatory region for thelupulin-specific gene expression” is meant nucleic acid comprising aregulatory region containing the promoter specifically functioning inlupulin glands. The nucleic acid can be isolated by the existing methodusing the reverse PCR with the DNA sequence of lupulin-specific cDNA asthe primer, for example, methods described in “Protocols for plant PCRexperiment” (Shu-jun Sha, p. 69 (1995)), incorporated herein byreference.

[0049] (7) DNA Sequencing

[0050] DNA sequence thus isolated can be determined by the existingmethod, for example, according to the protocol attached to an “ABI PRISMDye Primer Cycle Sequencing Ready Reaction Kit” (Perkin-Elmer),incorporated herein by reference. The DNA sequence thus decided can beexamined for the homology to that of existing genes in other plantspecies.

[0051] (8) Northern Hybridization Analysis (Hereinafter Referred to asNorthern Analysis)

[0052] Whether the lupulin-specific gene thus isolated is actuallyexpressed specifically in lupulin glands and whether the nucleic acidthus isolated comprising the lupulin-specific expression regulatoryregion regulating the gene actually functions specifically in lupulinglands can be confirmed by carrying out Northern analysis with theisolated lupulin-specific gene as the probe. Northern analysis can beperformed by the existing method, for example, based on the methodsdescribed in “Protocols for non-isotope experiments-DIG hybridization(Shu-Jun Sha, p. 45 (1994)) and “User's guide for performing thehybridization using DIG system” (Boehringer Mannheim, p. 40 (1995)),both incorporated herein by reference.

[0053] 2. Preparation of Vectors Bearing the Above-isolatedLupulin-specific Gene or Lupuln-specific Expression Regulatory Sequence

[0054] Lupulin-specific gene the specificity of which in lupulin glandshas been confirmed as described above can be expressed, according to theexisting method, by inserting the gene downstream of the expressionregulatory sequence in a suitable vector bearing the expressionregulatory sequence followed by transferring the transformed vector toappropriate cells. There are no limitations on the type of vectorsbearing the expression regulatory sequence, and a vector described belowbearing lupulin-specific expression regulatory sequence and acommercially available expression vector (for example, pBI121 (CLONTECH)can be used.

[0055] Also, the construction of vector bearing the lupulin-specificexpression regulatory sequence can be similarly achieved by selecting anappropriate vector from existing plasmids according to the purpose andinserting the above-described expression regulatory sequence, forexample, SEQ ID NO:7 to it. In this case, the cloning region havingvarious restriction sites for linking structural genes may be optionallyincluded downstream of the expression regulatory sequence.

[0056] 3. Applications

[0057] Since secondary metabolic products are abundantly secreted in hoplupulin glands, the lupulin-specific genes isolated above are highlylikely to be the gene related to the biosynthesis of the secondarymetabolic products. Therefore, the application of genes obtained aboveto the transformation technique and marker assisted selection enables,for example, hop breeding based on the improvement of secondarymetabolic products formed in lupulin glands. For the above-describedtransformation technique, well-known methods can be used.

[0058] Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

EXAMPLES Example 1

[0059] Preparation of Lupulin-rich and Lupulin-poor Fractions

[0060] Hop cones were harvested 15 days after blooming, and frozen inliquid nitrogen. These frozen cones were dissected on dry-ice, anddivided using a dissection forceps into a fraction comprising mainlylupulin glands and the endocyte base with dense lupulin glands, and afraction comprising mainly the endocyst with few lupulin glands. Thesefractions were referred to as the lupulin-rich fraction and lupulin-poorfraction, respectively, and stored at −80° C.

Example 2

[0061] Preparation of Total RNA and mRNA from Lupulin-rich andLupulin-poor Fractions

[0062] Total RNA and mRNA of lupulin-rich fraction and lupulin-poorfraction were prepared as follows. Each fraction was frozen andpulferized in liquid nitrogen, suspended in a 2% CTAB solution(consisting of 2% cetyltrimethylammonium bromide, 0.1 M Tris (pH 9.5),20 mM EDTA, 1.4 M NaCl and 1% β-mercaptoethanol), and incubated at 65for 30 min. After the suspension was extracted twice withchloroform/isoamyl alcohol (24:1), a three quarters volume ofisopropanol was added to the extract to precipitate DNA and RNA. AfterDNA and RNA thus precipitated were dissolved in water, a {fraction(1/3)} volume of 10 M lithium chloride was added, and the resultingmixture was allowed to stand at −20° C. overnight, and then centrifugedat 15,000 rpm for 10 min. Precipitates thus obtained were washed with70% ethanol and dissolved in a DNase reaction buffer (consisting of 100mM sodium acetate (pH 5.2) and 5 mM magnesium chloride). To this mixturewas added DNase, and the resulting mixture was incubated at 37° C. todecompose DNA. To the incubation mixture was added a {fraction (1/3)}volume of 10 M lithium chloride, and the mixture was allowed to stand at−20° C. overnight, then centrifuged at 15,000 rpm for 10 min.Precipitates thus obtained were dissolved in water, and the resultingsolution was purified by the extraction with phenol-chloroform, andsubjected to the ethanol precipitation. Precipitates thus obtained weredissolved in water and used as the total RNA preparation, from whichmRNA was prepared using an “Oligotex-dT30<Super>” (Takara-Shuzo)according to the protocol attached thereto.

Example 3

[0063] Preparation of Lupulin-specific Probes

[0064] Herein, lupulin-specific probes were prepared by subtracting mRNApresent also in the lupulin-poor fraction from mRNA in the lupulin-richfraction. More specifically, the lupulin-specific probes were preparedusing a “Subtractor Kit” (Invitrogen) and according to the protocolattached to the Kit.

[0065] First, cDNA was synthesized from mRNA of the lupulin-richfraction. Also, mRNA in the lupulin-poor fraction was labelled withbiotin. Then, cDNA of the lupulin-rich fraction thus prepared was mixedwith the biotinized mRNA in the lupulin-poor fraction to form a hybrid,to which was then added streptoavidin to combine with the biotinizedmRNA in the hybrid. Then, the removal of the biotinized mRNA byextracting the hybrid with phenol-chloroform resulted in the depletionof cDNA derived from mRNA present also in the lupulin-poor fraction fromthe cDNA of the lupulin-rich fraction. As a result, cDNA derived onlyfrom the lupulin-rich fraction was obtained to be used as the probe.Lupulin-specific probes thus obtained were labelled with digoxigeninusing a “DIG-High Prime” (Boehringer Mannheim).

Example 4

[0066] Isolation of Lupulin-specific cDNA

[0067] A cDNA library was prepared from mRNA in the lupulin-richfraction using a “cDNA synthesis module”, “cDNA rapid adaptor ligationmodule” and “cDNA rapid cloning module—MOSSlox” (Amersham), and“GIGAPACK Plus Packaging Extract” (Stratagene) with—MOSSlox as thevector. This library was screened by the hybridization method usinglupulin-specific probes labelled with digoxigenin.

[0068] More specifically, each plaque derived from the above-describedcDNA library was transferred to membrane filter, and blocked in ahybridization buffer (containing 5×SSC, 100 mM phosphate buffer, 7% SDS,2% blocking agent, 0.1% N-lauroyl sarcosine, 50% formamide and 50 g/mlfish sperm DNA). Then, to the hybridization buffer was added theabove-described probe, and the membrane was incubated in the resultingmixture at 42° C. overnight. Then, the membrane was washed twice with arinsing solution (containing 1% SDS and 2×SSC) at 56° C. for 5 min, andfurther twice with a rinsing solution (containing 0.1% SDS and 0.1×SSC)at 68° C. for 5 min. Then, by detecting the positive plaque, thelupulin-specific cDNA was isolated.

Example 5

[0069] Determination of DNA Sequence of Lupulin-specific cDNA and AminoAcid Sequence of Translation Products

[0070] The DNA sequence of gene fragments containing thelupulin-specific cDNA and lupulin-specific promoter thus obtained wasthen determined. For sequencing, each gene fragment was subcloned intothe pUC vector or pBluescript vector. Sequencing was performed using a“ABI PRISM Dye Primer Cycle Sequencing Ready Reaction Kit” and a DNAsequencer (ABI373S model) (Perkin-Elmer).

[0071] The DNA sequence of the lupulin-specific cDNA thus determined isshown in SEQ ID NO:2. Also, the putative amino acid sequence of thetranslation product derived from the DNA sequence is shown in SEQ IDNO:1. In this case, the amino acid sequence of SEQ ID NO:1 correspondsto the DNA sequence from the initiation codon (ATG), position 36-38 tothe termination codon (TAA), position 1218-1220 in SEQ ID NO:2.

Example 6

[0072] Preparation of Hop Genomic DNA

[0073] Hop genomic DNA was prepared as follows. Leaves or cones of hopswere frozen and pulferized in liquid nitrogen, suspended in a 2% CTABsolution (containing 2% cetyltrimethylammonium bromide, 0.1 M Tris (pH9.5), 20 mM EDTA, 1.4 M NaCl and 1% β-mercaptoethanol), and incubated at65° C. for 30 min. The suspension was extracted twice withchloroform-isoamylalcohol (24:1), and added with a {fraction (3/4)}volume of isopropanol to precipitate DNA and RNA. DNA and RNA thusprecipitated were dissolved in a high salt TE buffer (containing 1 MNaCl, 10 mM Tris (pH 8.0) and 1 mM EDTA), added with RNase, and themixture was incubated at 60° C. to decompose RNA. To the reactionmixture was added 2 volumes of isopropanol to precipitate DNA, which waswashed with 70% ethanol, and then dissolved in water to obtain a genomicDNA sample.

Example 7

[0074] Isolation of the Expression Regulatory Sequence for theLupulin-specific Gene

[0075] Isolation of the expression regulatory sequence for thelupulin-specific gene was carried out using the inverse PCR method asfollows. FIG. 1 is a diagram representing the procedures in this Example7.

[0076] First, hop genomic DNA obtained in Example 6 was digested with arestriction enzyme Xho I (S1 and S2). Xho I digests were subjected toself circularization according to the protocol attached to a “DNALigation Kit Ver. 1” (Takara-Shuzo) (S3).

[0077] Next, the flanking region containing the promoter forlupulin-specific gene was amplified by PCR using primers having thesequence within the lupulin-specific cDNA with a portion of thisligation reaction mixture as the template (S4). Sequences of a pair ofprimers herein used are represented in SEQ ID NO:3 (primer 1) and NO:4(primer 2). Herein, SEQ ID NO:3 is a sequence complementary to that fromposition 137 to 166 and SEQ ID NO:4 is a sequence from position 303 to332 of SEQ ID NO:2, respectively.

[0078] The above-described PCR was performed using an “ExpandHigh-Fidelity PCR System” (Boehringer-Mannheim) according to theprotocol attached thereto, incorporated herein by reference. Thereaction conditions were as follows: after 30 cycles of the incubationsat 94° C. for 1 min, 55° C. for 1 min and 68° C. for 4 min, the mixturewas further incubated at 72° C. for 6 min.

[0079] The reaction solution thus obtained was electrophoresed for theidentification of PCR products. Since, in addition to the amplifiedfragment 1, non-specific amplified fragments might be contained in theabove PCR products, a selective amplification of only the desiredfragment was further attempted using different primers (S5). That is, inorder to selectively amplify only DNA segment comprising thelupulin-specific promoter, PCR was performed with a portion of theabove-described PCR solution as the template using primer 3 (SEQ IDNO:5) complementary to the sequence further upstream of thelupulin-specific gene than primer 1 and the above-described primer 2(S6). The primer 3 (SEQ ID NO:5) comprises the sequence complementary tothat from Nos. 114 to 143 of the lupulin-specific cDNA (SEQ ID NO:2).PCR was carried out using the same conditions and apparatus as describedabove. Then, the DNA sequence was determined using the PCR-amplifiedfragment obtained using these primers 2 and 3.

Example 8

[0080] DNA Sequence Determination of the Expression Regulatory Sequencefor the Lupulin-specific Gene

[0081] Base sequence of the above-described amplified fragment 2 wasdetermined similarly as in Example 5 described above by subcloning theamplified fragment to the pUC vector or pBluescript vector and using anABI PRISM Dye Primer Cycle Sequencing Ready Reaction Kit and a DNAsequencer (ABI373S type) (Perkin-Elmer). Results are shown in SEQ IDNO:6.

[0082] Since this amplified fragment was obtained by the inverse PCRmethod, it is expected that the amplified fragment contains theexpression regulatory sequence such as that of the promoter within thelupulin-specific gene (a portion thereof). Therefore, in order toidentify this expression regulatory sequence, the DNA fragment thusamplified was compared with DNA sequence of the lupulin-specific cDNA.These comparisons revealed that the DNA fragment herein amplified (SEQID NO:6) contained the promoter sequence in the lupulin-specific cDNA.More specifically, the sequence position 1-690 of SEQ ID NO:6corresponded to the sequence position 303-992 of the lupulin-specificcDNA (SEQ ID NO:2), and the sequence position 3296-3438 of SEQ ID NO:6corresponded to the sequence Nos. 1-143 of the lupulin-specific cDNA(SEQ ID NO:2), respectively. Therefore, it has been indicated that theexpression regulatory region such as the promoter for thelupulin-specific gene is included in the region position 691-3295 of SEQID NO: 6, which is shown in SEQ ID NO:7.

Example 9

[0083] Northern Blot Analysis of the Lupulin-specific cDNA and theExpression Regulatory Sequence of the Lupulin-specific Gene

[0084] Whether the above-described lupulin-specific cDNA was actuallyexpressed in lupulin glands and whether the promoter for theabove-described lupulin-specific gene actually functioned in lupulinglands were examined by the Northern blot analysis for the total RNAsextracted from the lupulin-rich and lupulin-poor fractions,respectively, using labelled DNAs prepared based on the lupulin-specificcDNA (SEQ ID NO:2) in Example 5.

[0085] First, the total RNAs from the lupulin-rich and lupulin-poorfractions were prepared by a similar method as in Example 2, andfractionated by denaturing agarose gel electrophoresis (1% agarose, 18%formaldehyde, 20 mM MOPS, 5 mM sodium acetate and 1 mM EDTA (pH 7)).RNAs thus fractionated in the agarose gel were transferred to nylonmembrane, and subjected to hybridization using cDNA obtained above asthe probe according to the Users guide for hybridization with DIG System(Boehringer-Mannheim, p.40 (1995)), incorporated herein by reference.

[0086] Hybridization was performed under the following conditions. Theabove-described membrane was blocked using a hybridization buffer of thesame constituents as in Example 4. To the above-described hybridizationbuffer was added the lupulin-specific cDNA labelled with digoxigenin asthe probe, the blocked membrane was soaked into this mixture, andincubated at 50° C. overnight. Then, the membrane was rinsed twice at56° C. for 10 min with a washing solution (containing 1% SDS and 2×SSC),further twice at 68° C. for 30 min with a washing solution (containing0.1% SDS and 0.1×SSC), and then searched for bands fused with the probe.Results are shown in FIG. 2.

[0087] As represented in FIG. 2, although a few mRNAs for the geneobtained above were also present in the lupulin-poor fraction, they wereclearly present in abundance in the lupulin-rich fraction, indicatingthe strong expression of this gene specifically in lupulin glands. Theexpression of this gene is controlled by the nucleic acid comprising theexpression regulatory region containing the promoter localized upstreamof the structural gene in the genomic DNA, and the nucleic acidcomprising the expression regulatory region is the one isolated andidentified in the above-described example, indicating a specificallystrong function of the above-described isolated nucleic acid comprisingthe expression regulatory region in lupulin glands. Signal bandsdetected in the low molecular side were thought to be decomposedproducts of mRNA of the gene obtained above.

Example 10

[0088] Homology Examination

[0089] The putative amino acid sequence derived from the DNA sequence ofthe lupulin-specific cDNA thus obtained was compared for homology withthe existing amino acid sequences. As a result, the gene had a highhomology with the gene for chalcone synthetase catalyzing the synthesisof nalingenin concerned to the biosynthesis of flavonoids in plants.More specifically, in comparison with chalcon synthetases from otherplants such as Arabidopsis (Plant J., 8 (5), 659-671 (1995)), barley(Plant Mol. Biol. 16:1103-1106 (1991)), pea (EMBL/GenBank/DDBJ databasesX80007), petunia (J. Biotechnol., 11 (2), 131-135 (1995) and rye(EMBL/GenBank/DDBJ databases X92547), the hop enzyme showed 65-70%homology in the DNA level and 70-75% homology in the amino acid level.

[0090] Recently, chalcone synthetase has been indicated to have theactivity of valerophenone synthetase catalyzing phlorisovalerophenoneand phlorisobutylophenone, precursors of bitter substance, α-acid andβ-acid (European Brewery Convention, Proceedings of the 26th Congress,p. 215 (1997)), indicating a possibility for the translation product ofthe gene obtained above to participate in the biosynthesis of bittersubstance as valerophenone synthetase.

[0091] Therefore, in the event that the gene specifically expressed inlupulin glands encodes chalcone synthetase, this nucleic acid can beused for improving flavonoids in plants. Also, in the case that thisgene encodes valerophenone synthetase, this nucleic acid can be used forimproving bitter substance in hops. Furthermore, since hop bittersubstance, α-acid and β-acid, have pharmacological activity (Biosci.Biotech. Biochem. 61 (1), 158 (1997)), it is possible for theabove-described nucleic acid to be applied to drug production.

[0092] Industrial Applicability

[0093] As described above, nucleic acids comprising genes specificallyexpressed in hop lupulin glands enable the breeding of hops by geneticengineering techniques focused on secondary metabolic products expressedin lupulin glands. Also, in the case of the use of vectors bearing theabove-described lupulin-specific genes, it is expected that theproduction of secondary metabolic products can be achieved outside ofplants, such as in cultured cells. Since such secondary metabolicproducts include important materials such as foods and drugs, and alsosince chalcone synthetase is involved in the biosynthesis of flavonoids,and valerophenone synthetase is involved in the biosynthesis of bittersubstance, the present invention is expected to greatly contribute tothe development and improvement of materials for foods and medicines.

[0094] Furthermore, the lupulin-specific promoter in the presentinvention can be utilized for the improvement of secondary metabolicproducts such as essential oil constituents and bitter substanceaccumulated in lupulin glands by inserting the gene of interestdownstream of the promoter. The promoter can also be used forintroducing novel other characters to hops.

[0095] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

[0096] This application is based on Japanese Patent Application Ser. No.Hei 10-37266, filed on filed on Feb. 19, 1998, and Japanese PatentApplication Ser. No. Hei 10-174235, filed on filed on Jun. 22, 1998,both of which are incorporated herein by reference in their entirety.

1 7 1 394 PRT Humulus lupulus 1 Met Ala Ser Val Thr Val Glu Gln Ile ArgLys Ala Gln Arg Ala Glu 1 5 10 15 Gly Pro Ala Thr Ile Leu Ala Ile GlyThr Ala Val Pro Ala Asn Cys 20 25 30 Phe Asn Gln Ala Asp Phe Pro Asp TyrTyr Phe Arg Val Thr Lys Ser 35 40 45 Glu His Met Thr Asp Leu Lys Lys LysPhe Gln Arg Met Cys Glu Lys 50 55 60 Ser Thr Ile Lys Lys Arg Tyr Leu HisLeu Thr Glu Glu His Leu Lys 65 70 75 80 Gln Asn Pro His Leu Cys Glu TyrAsn Ala Pro Ser Leu Asn Thr Arg 85 90 95 Gln Asp Met Leu Val Val Glu ValPro Lys Leu Gly Lys Glu Ala Ala 100 105 110 Ile Asn Ala Ile Lys Glu TrpGly Gln Pro Lys Ser Lys Ile Thr His 115 120 125 Leu Ile Phe Cys Thr GlySer Ser Ile Asp Met Pro Gly Ala Asp Tyr 130 135 140 Gln Cys Ala Lys LeuLeu Gly Leu Arg Pro Ser Val Lys Arg Val Met 145 150 155 160 Leu Tyr GlnLeu Gly Cys Tyr Ala Gly Gly Lys Val Leu Arg Ile Ala 165 170 175 Lys AspIle Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Ile Val Cys 180 185 190 SerGlu Ile Thr Ala Cys Ile Phe Arg Gly Pro Ser Glu Lys His Leu 195 200 205Asp Cys Leu Val Gly Gln Ser Leu Phe Gly Asp Gly Ala Ser Ser Val 210 215220 Ile Val Gly Ala Asp Pro Asp Ala Ser Val Gly Glu Arg Pro Ile Phe 225230 235 240 Glu Leu Val Ser Ala Ala Gln Thr Ile Leu Pro Asn Ser Asp GlyAla 245 250 255 Ile Ala Gly His Val Thr Glu Ala Gly Leu Thr Phe His LeuLeu Arg 260 265 270 Asp Val Pro Gly Leu Ile Ser Gln Asn Ile Glu Lys SerLeu Ile Glu 275 280 285 Ala Phe Thr Pro Ile Gly Ile Asn Asp Trp Asn AsnIle Phe Trp Ile 290 295 300 Ala His Pro Gly Gly Pro Ala Ile Leu Asp GluIle Glu Ala Lys Leu 305 310 315 320 Glu Leu Lys Lys Glu Lys Met Lys AlaSer Arg Glu Met Leu Ser Glu 325 330 335 Tyr Gly Asn Met Ser Cys Ala SerVal Phe Phe Ile Val Asp Glu Met 340 345 350 Arg Lys Gln Ser Ser Lys GluGly Lys Ser Thr Thr Gly Asp Gly Leu 355 360 365 Glu Trp Gly Ala Leu PheGly Phe Gly Pro Gly Leu Thr Val Glu Thr 370 375 380 Val Val Leu His SerVal Pro Thr Asn Val 385 390 2 1359 DNA Humulus lupulus 2 tttcacagtactactagcta tatatatatc aggtaatggc gtccgtaact gtagagcaaa 60 tccgaaaggctcagcgagct gaaggtccgg ccaccatcct cgccattggc accgccgttc 120 ctgccaactgtttcaaccaa gctgattttc ccgactacta ctttcgtgtc accaaaagtg 180 aacacatgactgatctcaaa aagaagttcc aacgaatgtg tgaaaaatcc actataaaaa 240 agcgttacttgcacttgacc gaagagcatc tgaagcagaa cccacatctg tgcgagtaca 300 atgcaccatctctgaacaca cgccaagaca tgttggtggt tgaagttccc aagcttggga 360 aggaggctgcaatcaatgcc atcaaagaat ggggccaacc caagtccaag atcacccatc 420 tcatcttctgcaccggctcc tccatcgaca tgccaggagc cgattaccaa tgcgccaagc 480 ttctcggcctccgaccctcg gtgaagcgag tgatgctgta tcaactcggc tgttatgccg 540 gtggaaaagttcttcgcata gccaaggaca tagcagagaa caacaagggc gctagagttc 600 tcattgtgtgctctgagatc acagcttgta tctttcgcgg gccctcggag aaacatttgg 660 attgcttggtggggcaatct ctgttcggag acggggcatc ttcggtcatc gttggtgccg 720 accctgatgcctcggtaggc gagcggccga tcttcgagtt ggtttcagct gcgcagacga 780 ttttgcctaactcggatgga gccatagccg ggcacgtaac ggaagccggg ctgacatttc 840 acttgctgagggacgtgcca gggttgatct cccaaaacat tgagaagagc ttgattgagg 900 ccttcactccgattgggatt aatgactgga acaacatatt ctggattgca catcccggtg 960 gacctgccattctggacgag atagaggcca agctcgagct gaagaaggag aagatgaagg 1020 cgtctcgtgaaatgctgagc gagtatggga acatgtcatg tgcaagcgtt ttcttcatag 1080 tagatgagatgaggaaacag tcgtcgaagg aagggaagtc taccaccgga gatggactgg 1140 agtggggcgctctcttcggg tttggaccgg gtctgacggt ggagacggtg gtcttgcaca 1200 gcgtgcccacaaacgtctaa tgaataattt gttatcgcta gcttgtcaaa tcaagcttta 1260 ctatgtattgtggtcgttaa ttagtttata ctttgatgtt gatcaataat tatatacctc 1320 atctaataaaatgatcaaat atatttttat ataaaaaaa 1359 3 30 DNA Artificial Sequencepolynucleotide 3 cgaaagtagt agtcgggaaa atcagcttgg 30 4 30 DNA ArtificialSequence polynucleotide 4 gcaccatctc tgaacacacg ccaagacatg 30 5 30 DNAArtificial Sequence polynucleotide 5 agcttggttg aaacagttgg caggaacggc 306 3439 DNA Humulus lupulus 6 gcaccatctc tgaacacacg ccaagacatg ttggtggttgaagttcccaa gcttgggaag 60 gaggctgcaa tcaatgccat caaagaatgg ggccaacccaagtccaagat cacccatctc 120 atcttctgca ccggctcctc catcgacatg ccaggagccgattaccaatg cgccaagctt 180 ctcggcctcc gaccctcggt gaagcgagtg atgctgtatcaactcggctg ttatgccggt 240 ggaaaagttc ttcgcatagc caaggacata gcagagaacaacaagggcgc tagagttctc 300 attgtgtgct ctgagatcac agcttgtatc tttcgcgggccctcggagaa acatttggat 360 tgcttggtgg ggcaatctct gttcggagac ggggcatcttcggtcatcgt tggtgccgac 420 cctgatgcct cggtaggcga gcggccgatc ttcgagttggtttcagctgc gcagacgatt 480 ttgcctaact cggatggagc catagccggg cacgtaacggaagccgggct gacatttcac 540 ttgctgaggg acgtgccagg gttgatctcc caaaacattgagaagagctt gattgaggcc 600 ttcactccga ttgggattaa tgactggaac aacatattctggattgcaca tcccggtgga 660 cctgccattc tggacgagat agaggccaag ctcgaggagtttggagactg tccgaggtcc 720 ttctcctagg gtgatcacca gctcgatagt ccctatagccgttgatcctt ctcccgaaaa 780 accgcacagc atcatggagg tcgccttcag ctcggcgacagtcaaaccca tcttctccaa 840 cgtggaccgg aatagaaggt ttaccgagct cccattatcgatcaacaccc tcctaaccct 900 ccgaatagcg agctgaactg ctacgaccag agggtcgttatgagggaact ggacatggcc 960 cgcatcttct tctgtaaaaa tgatcggttg cctctccaatcgctgctgct ttggcagacg 1020 ctgctccggg acgaactcta ctccattatg cgccttgagttcgtttacgt atctcttttg 1080 ggcaccccta ctcacgctag ctaaatgcgg acctccagagattgtggata tctctcctcc 1140 aatcactgga ggagggacgt cttgatctat ccgagacccagaaatctata caaaaaaaaa 1200 actatgtata aggttcataa acacattata ttcattaatttaaccttaaa attaaaaaaa 1260 atgaaaaaaa ctcaccaaaa ttggtctagg aagtcggagacgccgctagt tcttgggaga 1320 aaacctaagt tttgaatttg ggagaatgaa gggcttggggtcgatggctg agatttaata 1380 ctgggtgcac tgtttgcgtt agtgggcaac tgacgctaacggcttgtttg catcagtgcc 1440 aaactgacgc aaacacaccg ttagcgttag ttgcccactgacgcaaacgg tgcattaaga 1500 gcatcagttg gccactgacg caaacttcac caattaacagtgtcagtgtt atcactgatg 1560 caaatgcccc tgaatttgtg gtagtactca acttccacaaatgctgattc tcggtcaacg 1620 gcgtcagtca actgtgttga gtgacgcgtt tgactgacacaaaataagta ttttggtgta 1680 gtggaagatt aactaagaag gtaaaattgg aggttattgttatcactcct tcatcattta 1740 taaaagtaga aatacgttcc atttaatata ctaaccaaccttgctgccac atatcccctg 1800 aaaaaaataa aacaacaaca acctttctac cataaaattaggcatatgat gatatataac 1860 ctaactataa cacaaaatta ggcatatgat gatatatataacctaactat aacacaaaat 1920 taggcatata tatatacact cacaaatagt ggctgctatacccaacacct taattaatta 1980 atagttaatg ctcctctaga agactggacg agatcaagtgctattatgcg gaatcaagat 2040 ctcctatcaa aaaaagatgt cccagcctat gtttagaaaatgttaaatca aattctgtta 2100 actaatttct atatttctca tccctactcc tttttttttaacaatcaaca attcattgaa 2160 aataatcaaa atgtaataca actaataata agatgatatatatagtaact atccatacaa 2220 gttcattatc cactctaagt gcatgcacaa ttcatgaacggccttattgg ccaaacgtca 2280 aacacaaatt agagatacct tagaaaaatt ggataataaacttgttatat tttctaacaa 2340 agaccctaat tcattactac tccattaaat gacgtgtatctttcattttt ttttaaaaat 2400 tttagaaact aatagagtat ggattgatgc tgcattataagaaatcgatc acaccttcag 2460 ttatgaactt tccggctaag caccatcggg catctatgtcctcctctttt gccacattat 2520 catatgaaat accactgttt tcctcctctt ccaagcttatggtcaagacc ggccctgaac 2580 taaggtgggt tagacccacg cctagggcct atttttttttacatttcttt taaaaatact 2640 ataaattttt aaaaagtttt tacaaaaagg gcccctaatcaccaattttt cctaaggctc 2700 aaaactcttc agggccagcc ctgcctatgg tagcatatctagattctaaa tcttgcttat 2760 gagaactgct cgatgccata acttccttcg ccaccaagactaataacaca aacaatagag 2820 aacgaacaca ccaatagcaa tacaaaacac cttacgtcaactgacccaac agagagctac 2880 catgtcaaaa gacaatacta gtttgagact tcaccactgtcaaaattcta gttctcaaca 2940 ctagcaaaaa aaaaagtgtt aaacaccatc aatcacataacgacatactt cttggccata 3000 ttttttttcc catgtaatca tgtaaaaggt ggggaaaataaatcaataca cataaagaac 3060 aatgaaaaaa taaataaaca agtcaaatta ttataatttaacattaataa aaagttgaga 3120 atcacaaaca ttggtacgta ggtattaggg ttggtgtttacacatattat ccataggcca 3180 tgcacacctt acctaaccca tgcaccactt tgtacatattatatatataa ctccaatttg 3240 gctttgcatt tcaacacttg taatcattac actatatttgtgtatatagt gtaagttttc 3300 acagtactac tagctatata tatatcaggt aatggcgtccgtaactgtag agcaaatccg 3360 aaaggctcag cgagctgaag gtccggccac catcctcgccattggcaccg ccgttcctgc 3420 caactgtttc aaccaagct 3439 7 2606 DNA Humuluslupulus 7 ctcgaggagt ttggagactg tccgaggtcc ttctcctagg gtgatcaccagctcgatagt 60 ccctatagcc gttgatcctt ctcccgaaaa accgcacagc atcatggaggtcgccttcag 120 ctcggcgaca gtcaaaccca tcttctccaa cgtggaccgg aatagaaggtttaccgagct 180 cccattatcg atcaacaccc tcctaaccct ccgaatagcg agctgaactgctacgaccag 240 agggtcgtta tgagggaact ggacatggcc cgcatcttct tctgtaaaaatgatcggttg 300 cctctccaat cgctgctgct ttggcagacg ctgctccggg acgaactctactccattatg 360 cgccttgagt tcgtttacgt atctcttttg ggcaccccta ctcacgctagctaaatgcgg 420 acctccagag attgtggata tctctcctcc aatcactgga ggagggacgtcttgatctat 480 ccgagaccca gaaatctata caaaaaaaaa actatgtata aggttcataaacacattata 540 ttcattaatt taaccttaaa attaaaaaaa atgaaaaaaa ctcaccaaaattggtctagg 600 aagtcggaga cgccgctagt tcttgggaga aaacctaagt tttgaatttgggagaatgaa 660 gggcttgggg tcgatggctg agatttaata ctgggtgcac tgtttgcgttagtgggcaac 720 tgacgctaac ggcttgtttg catcagtgcc aaactgacgc aaacacaccgttagcgttag 780 ttgcccactg acgcaaacgg tgcattaaga gcatcagttg gccactgacgcaaacttcac 840 caattaacag tgtcagtgtt atcactgatg caaatgcccc tgaatttgtggtagtactca 900 acttccacaa atgctgattc tcggtcaacg gcgtcagtca actgtgttgagtgacgcgtt 960 tgactgacac aaaataagta ttttggtgta gtggaagatt aactaagaaggtaaaattgg 1020 aggttattgt tatcactcct tcatcattta taaaagtaga aatacgttccatttaatata 1080 ctaaccaacc ttgctgccac atatcccctg aaaaaaataa aacaacaacaacctttctac 1140 cataaaatta ggcatatgat gatatataac ctaactataa cacaaaattaggcatatgat 1200 gatatatata acctaactat aacacaaaat taggcatata tatatacactcacaaatagt 1260 ggctgctata cccaacacct taattaatta atagttaatg ctcctctagaagactggacg 1320 agatcaagtg ctattatgcg gaatcaagat ctcctatcaa aaaaagatgtcccagcctat 1380 gtttagaaaa tgttaaatca aattctgtta actaatttct atatttctcatccctactcc 1440 ttttttttta acaatcaaca attcattgaa aataatcaaa atgtaatacaactaataata 1500 agatgatata tatagtaact atccatacaa gttcattatc cactctaagtgcatgcacaa 1560 ttcatgaacg gccttattgg ccaaacgtca aacacaaatt agagataccttagaaaaatt 1620 ggataataaa cttgttatat tttctaacaa agaccctaat tcattactactccattaaat 1680 gacgtgtatc tttcattttt ttttaaaaat tttagaaact aatagagtatggattgatgc 1740 tgcattataa gaaatcgatc acaccttcag ttatgaactt tccggctaagcaccatcggg 1800 catctatgtc ctcctctttt gccacattat catatgaaat accactgttttcctcctctt 1860 ccaagcttat ggtcaagacc ggccctgaac taaggtgggt tagacccacgcctagggcct 1920 attttttttt acatttcttt taaaaatact ataaattttt aaaaagtttttacaaaaagg 1980 gcccctaatc accaattttt cctaaggctc aaaactcttc agggccagccctgcctatgg 2040 tagcatatct agattctaaa tcttgcttat gagaactgct cgatgccataacttccttcg 2100 ccaccaagac taataacaca aacaatagag aacgaacaca ccaatagcaatacaaaacac 2160 cttacgtcaa ctgacccaac agagagctac catgtcaaaa gacaatactagtttgagact 2220 tcaccactgt caaaattcta gttctcaaca ctagcaaaaa aaaaagtgttaaacaccatc 2280 aatcacataa cgacatactt cttggccata ttttttttcc catgtaatcatgtaaaaggt 2340 ggggaaaata aatcaataca cataaagaac aatgaaaaaa taaataaacaagtcaaatta 2400 ttataattta acattaataa aaagttgaga atcacaaaca ttggtacgtaggtattaggg 2460 ttggtgttta cacatattat ccataggcca tgcacacctt acctaacccatgcaccactt 2520 tgtacatatt atatatataa ctccaatttg gctttgcatt tcaacacttgtaatcattac 2580 actatatttg tgtatatagt gtaagt 2606

1. An isolated and purified nucleic acid comprising a gene specificallyexpressed in hop lupulin glands or a portion thereof.
 2. The nucleicacid of claim 1, wherein the gene encodes at least one protein involvedin the biosynthesis of secondary metabolic products.
 3. The nucleic acidof claim 2, wherein the protein encoded by the nucleic acid comprisesthe amino acid sequence of SEQ ID NO:
 1. 4. The nucleic acid of claim 1,wherein the gene comprises the DNA sequence of SEQ ID NO:
 2. 5. Thenucleic acid of claim 1, wherein the gene hybridizes with the DNAsequence of SEQ ID NO: 2 or a portion thereof.
 6. The nucleic acid ofclaim 2, wherein the protein encoded by the nucleic acid is a chalconesynthetase.
 7. A vector comprising the nucleic acid of claim
 1. 8. Anisolated and purified nucleic acid comprising a regulatory sequence forthe specific expression of genes in hop lupulin glands.
 9. The nucleicacid of claim 8, wherein the regulatory sequence comprises a promoter.10. The nucleic acid of claim 8, wherein the regulatory sequencecomprises the DNA sequence of SEQ ID NO:
 7. 11. The nucleic acid ofclaim 8, wherein the regulatory sequence comprises the DNA sequence ofSEQ ID NO: 6 or a portion thereof.
 12. A vector comprising the nucleicacid of claim
 8. 13. A plant cell transformed by the vector of claim 7.14. A plant cell transformed by the vector of claim
 12. 15. A method ofproducing a transformed plant cell, comprising transforming a plant cellwith the vector of claim
 7. 16. A method of producing a transformedplant cell, comprising transforming a plant cell with the vector ofclaim
 12. 17. A nucleic acid primer comprising the DNA sequence of SEQID NO: 3, 4 or
 5. 18. A kit for detecting regulatory sequences for thespecific expression of lupulin-specific expression genes, comprising atleast one nucleic acid having the base sequence of SEQ ID NOs: 3, 4 or5.